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Listeriosis is currently the fifth most common foodborne disease in Europe. Most cases are sporadic; however, outbreaks have also been reported. Compared to other foodborne infections, listeriosis has a modest incidence but can cause life-threatening complications, especially in elderly or immunocompromised people and pregnant women. In the latter case, the pathology can be the cause of premature birth or spontaneous abortion, especially if the fetus is affected during the first months of gestation. The causative agent of listeriosis, Listeria monocytogenes, is characterized by the innate ability to survive in the environment and in food, even in adverse conditions and for long periods. Ready-to-eat food represents the category most at risk for contracting listeriosis. This study presents the result of an investigation carried out on a case of maternal-fetal transmission of listeriosis which occurred in 2020 in central Italy and which was linked, with a retrospective approach, to other cases residing in the same city of the pregnant woman. Thanks to the use of next-generation sequencing methodologies, it was possible to identify an outbreak of infection, linked to the consumption of ready-to-eat sliced products sold in a supermarket in the investigated city.

Salmonella is a Gram-negative enteric bacterium responsible for the foodborne and waterborne disease salmonellosis, which is the second most reported bacterial zoonosis in humans. Many animals are potential sources of salmonellosis, including dogs, cats, and other pets. We report the case of an outbreak of salmonellosis in a family in central Italy, affecting two children and involving their three dogs as carriers. One of the children needed medical care and hospitalisation. Isolation and analysis of stool samples from the sibling and the animals present in the house were carried out. Serotyping allowed the identification of S. enterica subsp. enterica serovar Typhimurium in its monophasic variant for all the isolates. The results of whole-genome sequencing confirmed that the strains were tightly related. The minimum inhibitory concentration (MIC) test documented the resistance to ampicillin, sulfamethoxazole, and tetracycline. The origin of the zoonotic outbreak could not be assessed; however, the case study showed a clear passage of the pathogen between the human and non-human members of the family. The possibility of a transmission from a dog to a human suggests the need for further studies on the potential ways of transmission of salmonellosis through standard and alternative feed.

Hepatitis E is an emerging disease in industrialized countries. The food-borne transmission of hepatitis E virus (HEV) is associated principally with products derived from the domestic pig, the wild boar, and deer; however, few quantitative data are available on HEV loads in animals used in food production. This study assessed HEV occurrence, viral load and genetic variability in wild boar hunted for domestic consumption in the district of Viterbo (Central Italy) where high anti-HEV IgG seroprevalence values are reported in humans. A total of 332 liver and 69 intestine samples were obtained from wild boar hunted between 2011 and 2014. The liver tissue in 54 of the animals (16.3%) was HEV-positive. Viral loads in quantifiable liver samples (n = 29) ranged between 3.2 × 102 and 3.8 × 105 genome copies (g.c.)/g with a mean value of 1.85 × 104 g.c./g. A statistically significant positive correlation was found between viral concentration in liver and intestinal tissue, though mean viral load in the intestines was lower (3.13 × 103 g.c./g). Twenty-six samples were characterized molecularly as genotype 3 (G3) and four subtypes (a, c, f and l) were detected. Finally, twelve samples with near identical sequences were identified as G3 but could not be assigned to any of the known subtypes, and could therefore represent a potentially new subtype.

Wild boar is an animal the population of which constantly increases in Europe. This animal plays an important role as a reservoir for several pathogens, including three of the most important zoonoses: salmonellosis, yersiniosis and listeriosis. The aim of this investigation was to evaluate the occurrence of antimicrobial-resistant and virulence factor genes of Salmonella spp., Yersinia enterocolitica and Listeria monocytogenes isolated from wild boar in Tuscany (Central Italy). During two consequent hunting seasons (2018/2019 and 2019/2020), rectal swabs, spleens and livers were collected from 287 hunted wild boar to isolate strains. Each isolate was tested to investigate its antimicrobial resistance and to detect virulence factor genes by PCR. Eighteen Salmonella strains (6.27%) were isolated. Of these, 66.7% were resistant to streptomycin, 13.4% to cephalothin, 6.67% to imipenem and one isolate (6.67%) was resistant simultaneously to five antimicrobials. Moreover, the most detected genes were sopE (73.4%), pipB (66.7%), sodCI (53.3%), spvR and spvC (46.7%). In total, 54 (17.8%) Yersinia enterocolitica were isolated; of them, 26 (48.1%), 9 (16.7%), 17 (31.5%), 1 (1.85%) and 1 (1.85%) belonged to biotypes 1, 2, 3, 4 and 5, respectively. All strains (100%) demonstrated resistance to cephalothin and 70.4% to amoxicillin-clavulanic acid, 55.6% to ampicillin, and 37.0% to cefoxitin. Additionally, the most detected genes were ystA (25.9%), inv (24.1%), ail (22.2%), ystB (18.5%) and virF (14.8%). Finally, only one Listeria monocytogenes isolate (0.35%) was obtained, belonging to serogroup IVb, serovar 4b, and it was found to be resistant to cefoxitin, cefotaxime and nalidixic acid. The results highlighted the role of wild boar as a carrier for pathogenic and antimicrobial-resistant Salmonella spp., Yersinia enterocolitica and Listeria monocytogens, representing a possible reservoir for domestic animals and human pathogens.

Listeria monocytogenes(Lm) is a ubiquitous bacterium that causes the foodborne illness, listeriosis. Clonal complexes (CC), such as CC121, are overrepresented in the food production industry, and are rarely reported in animals and the environment. Working within a European-wide project, we investigated the routes by which strains are transmitted from environments and animals to food and the food production environment (FPE). In this context, we report, for the first time, the occurrence of a ST121 (CC121) strain isolated from a dolphin brain. The genome was compared with the genomes of 376 CC121 strains. Genomic comparisons showed that 16 strains isolated from food were the closest to the dolphin strain. Like most of the food strains analyzed here, the dolphin strain included genomic features (transposon Tn6188, plasmid pLM6179), both described as being associated with the strain’s adaptation to the FPE. Like all 376 strains, the dolphin strain contained a truncatedactAgene andinlAgene, both described as being associated with attenuated virulence. Despite this fact, the strain was able to cross blood-brain barrier in immunosuppressed dolphin exposed polychlorinated biphenyl and invaded by parasites. Our data suggest that the dolphin was infected by a food-related strain released into the Mediterranean Sea.View Full-Text LISTADAPT

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes the foodborne illness, listeriosis. Clonal complexes (CC), such as CC121, are overrepresented in the food production industry, and are rarely reported in animals and the environment. Working within a European-wide project, we investigated the routes by which strains are transmitted from environments and animals to food and the food production environment (FPE). In this context, we report, for the first time, the occurrence of a ST121 (CC121) strain isolated from a dolphin brain. The genome was compared with the genomes of 376 CC121 strains. Genomic comparisons showed that 16 strains isolated from food were the closest to the dolphin strain. Like most of the food strains analyzed here, the dolphin strain included genomic features (transposon Tn6188, plasmid pLM6179), both described as being associated with the strain’s adaptation to the FPE. Like all 376 strains, the dolphin strain contained a truncated actA gene and inlA gene, both described as being associated with attenuated virulence. Despite this fact, the strain was able to cross blood-brain barrier in immunosuppressed dolphin exposed polychlorinated biphenyl and invaded by parasites. Our data suggest that the dolphin was infected by a food-related strain released into the Mediterranean Sea.

In the framework of a European Union (EU) Coordinated Monitoring Programme, different types of ready-to eat (RTE) products, including soft and semi-soft cheese (n = 398) and cooked meat products (n = 403), were collected at retail in Italy and tested for detection and enumeration of Listeria monocytogenes. An Integrative Survey of 2696 samples, including soft and semi-soft cheese (n = 894) and cooked meat products (n = 1802) was carried out to have statistically representative results at the national level. Considering the results obtained both from the EU and the national Integrative Survey, prevalence of contamination of meat products was 1.66% (95% CL: 1.02–2.73%) at the arrival of the samples at the laboratory and 1.92% (95% CL: 1.31%–2.82%) at the end of shelf-life. Spalla cotta was the most frequently contaminated meat product. Prevalence of contamination in cheese was 2.13% (95% CL: 1.37%–3.3%) at the arrival at the laboratory and 1.01% (95% CL: 0.41%–2.55%) at the end of shelf-life. To get information about differences between cheese rinds and pastes, these two parts were separately analysed in the Integrative Survey samples. L. monocytogenes was detected in 4.02% (95% CL: 2.60%–6.19%) of cheese rinds (n = 473), whereas only the 0.34% (95% CL: 0.12%–0.98%) of cheese pastes (n = 894) were contaminated. This difference was statistically significant (χ2 = 10.026, P

The aim of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli in fresh beef marketed in 2017 in 13 regions of Italy, to evaluate the potential risk to human health. According to the ISO/TS 13136:2012 standard, 239 samples were analysed and nine were STEC positive, from which 20 strains were isolated. The STEC-positive samples were obtained from Calabria (n = 1), Campania (n = 1), Lazio (n = 2), Liguria (n = 1), Lombardia (n = 1) and Veneto (n = 3). All STEC strains were analysed for serogroups O26, O45, O55, O91, O103, O104, O111, O113, O121, O128, O145, O146 and O157, using Real-Time PCR. Three serogroups were identified amongst the 20 strains: O91 (n = 5), O113 (n = 2), and O157 (n = 1); the O-group for each of the 12 remaining STEC strains was not identified. Six stx subtypes were detected: stx1a, stx1c, stx2a, stx2b, stx2c and stx2d. Subtype stx2c was the most common, followed by stx2d and stx2b. Subtype stx2a was identified in only one eae-negative strain and occurred in combination with stx1a, stx1c and stx2b. The presence in meat of STEC strains being potentially harmful to human health shows the importance, during harvest, of implementing additional measures to reduce contamination risk.

Non-typhoidal Salmonella enterica infection is a significant public health problem worldwide. The aim of this study was to characterize Salmonella enterica strains isolated from human specimens in central and southern Italy, for epidemiological studies. One hundred and fifty S. enterica strains were serotyped. Isolates were tested for their antimicrobial susceptibility, by disk diffusion method. The molecular characterizations, based on PCR, were carried out for the detection of invA gene and other virulence elements and phage marker genes. Eighteen different Salmonella serotypes were identified. The most common serotypes detected were S. Typhimurium, S. Enteritidis, the monophasic variant of S. Typhimurium (S. 4,[5],12:i:-), and S. Napoli. High resistance rates were recorded for tetracycline (64%), streptomycin (62%), sulphonamide (57%), and ampicillin (56%). The ASSuT R-type, also associated to resistance to other antibiotics, was highly prevalent in S. 4,[5],12:i:- (97%) and S. Typhimurium (55%), while the ACSSuT R-type, also associated to other antibiotics, was observed prevalently in S. Typhimurium (20.4%). The genes of more common detection were invA (100%), sspH2 (86.6%), gtgB (84.6%), g8 (80%), sodC1 (77.3%), gipA (52.6%), sspH1 (52.6%).

Prevalence data were collected from official microbiological records monitoring four selected foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7 and Campylobacter jejuni) in raw milk sold by self-service vending machines in seven Italian Regions (n. 60907 samples from 1239 vending machines) during the years 2008 to 2011. Data of samples analyzed both by culture-based and real-time PCR methods were collected in one Region. A total of 100 raw milk consumers in four regions were interviewed while purchasing raw milk from vending machines. One hundred and seventy eight samples out of 60907 were positive for one of the four foodborne pathogens investigated; overall, 18 samples were positive for Salmonella spp., 83 for L. monocytogenes, 24 for E. coli O157:H7 and 53 for C. jejuni in the seven Regions investigated. There were no significant differences in prevalence among Regions, but a significant increase in C. jejuni prevalence was observed over the years. A comparison of the two different analysis methods showed that real-time PCR is from 2.71 to 9.40 times more sensitive than culture-based method. Data on consumer habits showed that some behaviors may enhance the risk of infection due to raw milk consumption: 37% of consumers do not boil milk before consumption, 93% never use an insulated bag to transport raw milk home, and raw milk is consumed by children under five years of age. The study emphasizes that end-product controls alone are not sufficient to guarantee an adequate level of consumer protection. The beta distribution of positive samples in this study and the data on raw milk consumer habits are useful and appropriate for the development of a National Quantitative Risk Assessment of Salmonella spp., L. monocytogenes, E. coli O157 and C. jejuni related to raw milk consumption.

A quantitative risk assessment (RA) model was developed to describe the risk of campylobacteriosis linked to consumption of raw milk sold in vending machines in Italy. Exposure assessment was based on the official microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2011, microbial growth during storage, destruction experiments, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time-temperature field handling conditions detected. Two separate RA models were developed, one for the consumption of boiled milk and the other for the consumption of raw milk, and two different dose-response (D-R) relationships were considered. The RA model predicted no human campylobacteriosis cases per year either in the best (4°C) storage conditions or in the case of thermal abuse in case of boiling raw milk, whereas in case of raw milk consumption the annual estimated campylobacteriosis cases depend on the dose-response relationships used in the model (D-R I or D-R II), the milk time-temperature storage conditions, consumer behaviour and age of consumers, namely young (with two cut-off values of ≤5 or ≤6 years old for the sensitive population) versus adult consumers. The annual estimated cases for young consumers using D-R II for the sensitive population (≤5 years old) ranged between 1013.7/100,000 population and 8110.3/100,000 population and for adult consumers using D-R I between 79.4/100,000 population and 333.1/100,000 population. Quantification of the risks associated with raw milk consumption is necessary from a public health perspective and the proposed RA model represents a useful and flexible tool to perform future RAs based on local consumer habits to support decision-making on safety policies. Further educational programmes for raw milk consumers or potential raw milk consumers are required to encourage consumers to boil milk to reduce the associated risk of illness.

A quantitative risk assessment (RA) was developed to estimate haemolytic-uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7Âyears of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin-producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time–temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157-associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E.Âcoli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.

The aim of this study was to determine the prevalence of Shiga toxin-producing Escherichia coli in fresh beef marketed in 2017 in 13 regions of Italy, to evaluate the potential risk to human health. According to the ISO/TS 13136:2012 standard, 239 samples were analysed and nine were STEC positive, from which 20 strains were isolated. The STEC-positive samples were obtained from Calabria (n = 1), Campania (n = 1), Lazio (n = 2), Liguria (n = 1), Lombardia (n = 1) and Veneto (n = 3). All STEC strains were analysed for serogroups O26, O45, O55, O91, O103, O104, O111, O113, O121, O128, O145, O146 and O157, using Real-Time PCR. Three serogroups were identified amongst the 20 strains: O91 (n = 5), O113 (n = 2), and O157 (n = 1), the O-group for each of the 12 remaining STEC strains was not identified. Six stx subtypes were detected: stx1a, stx1c, stx2a, stx2b, stx2c and stx2d. Subtype stx2c was the most common, followed by stx2d and stx2b. Subtype stx2a was identified in only one eae-negative strain and occurred in combination with stx1a, stx1c and stx2b. The presence in meat of STEC strains being potentially harmful to human health shows the importance, during harvest, of implementing additional measures to reduce contamination risk.

The study summarises the results obtained over the period 2002-2013 by the Italian IT-Enter-Vet network, aimed at collecting data on Salmonella isolates from non-human sources. A total of 42,491 Salmonella isolates were reported with a progressive decrease over the years. S. Typhimurium was the most frequent serovar up to 2011, but then, it was overtaken by S. 4,[5],12,:i:-, S. Derby, S. Livingstone and S. Enteritidis alternated as the third most commonly isolated serovars. With regard to the sources of isolation, S. Typhimurium was distributed ubiquitously among the animal species. On the contrary, S. 4,[5],12,:i:- and S. Derby were strictly associated with pigs, whereas S. Livingstone, S. Enteritidis and S. Infantis were clearly related to poultry. Intriguingly, when the frequency of serovar distribution along the food chain was considered, it was evident that S. Typhimurium and S. Derby tended to persist along the chain, as they were isolated even more frequently from foods than from animals. A similar distribution was found for S. Enteritidis and S. Hadar. Despite limitations related to non-mandatory participation of laboratories in the network, the data presented are valuable to obtain a picture of the evolution of Salmonella from non-human sources over time in Italy.

The presence of foodborne pathogens (Salmonella spp., Listeria monocytogenes, Escherichia coli O157:H7, thermotolerant Campylobacter, Yersinia enterocolitica and norovirus) in fresh leafy (FL) and ready-to-eat (RTE) vegetable products, sampled at random on the Italian market, was investigated to evaluate the level of risk to consumers. Nine regional laboratories, representing 18 of the 20 regions of Italy and in which 97.7% of the country's population resides, were involved in this study. All laboratories used the same sampling procedures and analytical methods. The vegetable samples were screened using validated real-time PCR (RT-PCR) methods and standardized reference ISO culturing methods. The results show that 3.7% of 1372 fresh leafy vegetable products and 1.8% of 1160 "fresh-cut" or "ready-to-eat" (RTE) vegetable retailed in supermarkets or farm markets, were contaminated with one or more foodborne pathogens harmful to human health.

During the wild boars hunting season (2012-2013), 6508 adults were shot in Lazio Region, Viterbo territory. Out of the 6508, 393 subjects were sampled to be tested for the detection of Salmonella spp., Yersinia enterocolitica, E. coli VTEC and Trichinella spp. For this purpose, diaphragm and skeletal muscles samples were collected after the shooting, and tested at the Istituto Zooprofilattico Sperimentale Lazio and Toscana laboratories, Rome, Italy. Samples were tested respectively by polymerase chain reaction, enzyme linked immunofluorescent assay, microbiological and parasitological assays. The positivity percentages are 3.6% for Salmonella spp., 14.8% (out of 230 samples tested) for Yersinia enterocolitica, 4.6% for E.coli VTEC (vtx 1 and 2 genes). Each positive sample for vtx 1 and 2 was tested to isolate Verotoxigenic E.coli strains; none of them was positive for the same virulence genes. All diaphragm samples were negative to the trichinoscopic test. Every bacterial strain was identified serologically by seroagglutination test.

The authors describe a large outbreak of canine salmonellosis in a municipal kennel in Tuscany. During the outbreak, 174 samples of ‘diarrhetic’ and ‘normal’ faeces and two batches of commercial dehydrated dog food were cultured for pathogenic bacteria. The results of 25, out of a total of 41 dogs (60.9%) revealed at least one faecal sample as being positive for Salmonella; incidence per sampling ranged from 12.5% to 34%. Nine of 10 samples of dehydrated food were positive. Ten totally different serotypes were isolated from dry food and faeces: the results of the pulsed-field gel electrophoresis referred to similarity between the Salmonella Montevideo, Muenster and Worthington isolates recovered from both the food and canine faecal samples.